The overall goal of this project is to develop the reagents and culture conditions devoid of serum that will support the ex vivo expansion of hematopoietic stem cells (HSC) for short-term and long-term engraftment. The studies presented here will evaluate the culture conditions necessary to retain HSC capable of in vivo short-term and long-term repopulating activity using the recently development sheep in utero transplantation model for human hematopoiesis. Feasibility data presented in this proposal show that CD34+ cells from human bone marrow cultured in serum-free medium containing low levels of cytokines retained the long-term repopulating cells. In contrast, CD34+ cells cultured under similar conditions, but in the presence of serum, exhibited only short-term marrow repopulating activity. In the Phase I effort, the investigator proposes to extend these studies comparing the culture conditions devoid of serum with serum containing culture condition. The applicants propose to analyze the cell populations from each culture condition for 1) cell cycle status, 2) viable cell number, 3) GM-CFC, and 4) population dynamics of the more primitive HSC versus differentiation of the myeloid cells using immunophenotype analysis. Such information will be valuable in correlating the culture condition necessary to maintain and those that deplete the population of cells necessary for long-term engraftment. The Phase II proposal will be to expand these studies to include cord blood normal human bone marrow and mobilized peripheral blood stem cells and to develop and optimize the culture reagents and conditions devoid of serum, necessary for ex vivo expansion of the long- term engrafting cells. The Phase II effort will focus on cytokine mixtures, concentration effects and times of exposure to expand the long-term engrafting cells. Such studies will be available in utilizing ex vivo culture in clinical protocols for the treatment of disease, and will be invaluable in designing the clinical regimens for ex vivo culture of HSC for transplantation.